LOCUS       sequence-355468-        5527 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Trypanosoma brucei PCR only tagging (pPOT) vector for endogenous 
            gene tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5527)
  AUTHORS   Paterou A, Saez Conde J, Tyc J, Sunter JD, Vaughan S, Gull K, Dean S
  TITLE     A comprehensive toolkit for protein localization and functional 
            analysis in trypanosomatids.
  JOURNAL   Open Biol. 2025 Apr;15(4):240361. doi: 10.1098/rsob.240361. Epub 
            2025 Apr 2.
  PUBMED    40169015
REFERENCE   2  (bases 1 to 5527)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Open 
            Biol."; date: "2025-04"; volume: "15(4):240361. doi"; pages: " 
            10.1098/rsob.240361. Epub 2025 Apr 2"
COMMENT     Sequence Label: pPOTv7-blast-mNG
FEATURES             Location/Qualifiers
     source          1..5527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2652..2670)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2653..2670)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2688..2704)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2688..2704)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2701..2723)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2712..2728
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2736..2742,2743..2760,2761..2766))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2781..2802
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2919..2936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3090..3678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3170..3189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3849..4640,4641..4709))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVIIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4472..4491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4710..4814)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4892..5347)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5029..5050)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5241..5260
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5473..5495
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5487..5504
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5488..5504
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(5511..5527,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5511..5527,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgtata atgcagacct gctgcaagct aggggcacag caaggtcttc
       61 tgaaattcat gttttttttt tttttactct gcattgcagt ctccgctctt atttagtttt
      121 gctttacgta aggtctcgtt gctgccataa aataagctct agtggaattc atgcctttgt
      181 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
      241 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
      301 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
      361 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
      421 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga cctgcatcct gggatcaaag
      481 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
      541 ctggttatgt gtgggagggc taaccatggt gcccatttag ttggcctttc ccttgtctcg
      601 tgtcttttcc gtggaaaggt tcccggagta atctgatggc acagcaggga ggtgcgcctg
      661 caggttggtt aggaaggggg gatgatgtaa aagaagaaaa tggggggatt cgagccccac
      721 gcgttgctca agctgtgtag cgcaggcgtt tccttacata tttctctaac aggcacggaa
      781 gcctaacaaa tacacttggc ttattttttt gccccctcat gtcttgtaca aatatttgcg
      841 atagcttagc tatcagccac attaatcaaa caagtatacc aacaagcccg aaaacagaaa
      901 ctcaactgca acgggtacct atgggttctg gtagtggttc cggttccggt tctaagcttg
      961 tctcgaaagg tgaggaagac aatatggcca gcttgccagc aacgcacgag cttcatatat
     1021 ttgggtcgat caacggtgtg gatttcgaca tggtgggtca aggcacaggt aatcctaatg
     1081 acgggtatga agaactcaat ttgaagagca ctaagggaga tctccagttt tccccgtgga
     1141 ttcttgtgcc gcatattggt tatggttttc atcagtactt accatacccc gatggaatgt
     1201 cgcccttcca agcagctatg gtggacggct cggggtatca agtacatcgc accatgcaat
     1261 ttgaggacgg agcatccctc accgtaaact accgctatac gtatgagggg agccacataa
     1321 aaggtgaggc tcaagttaaa ggcacaggct tcccagctga cggcccggtc atgaccaatt
     1381 cattaaccgc tgctgattgg tgccgttcta agaagaccta ccctaacgac aagacgatca
     1441 ttagtacttt taagtggagt tataccacag gaaacggaaa gcggtatcga agcactgctc
     1501 gaacaaccta cacatttgcg aagccaatgg ccgcgaacta cttgaagaac cagccaatgt
     1561 atgtgtttcg aaaaacggaa ctgaagcatt cgaaaaccga gcttaatttt aaagagtggc
     1621 agaaggcgtt tacagacgtg atggggatgg acgaattgta taagggatcc ggatcaggat
     1681 ctggatcagg atcgggtagt taagagctcc gctgcgctta aatgtctttc attataatca
     1741 atgtataacc tttatgtagt atttcaatct atgccgctgt gtacgtgcac tgcggtgcct
     1801 atccttcggc attagagagt cactgtttgt gtagatcgta gctgcatgtc tgacatctgg
     1861 ttcattagtg ccgttttgag tctgcactgt tgtgcaactt gatattcatc actgcacacc
     1921 aaccgtgtgt ttcctccttt gggtgcaccc tttaatcctt gtcttctcct tttttgtctc
     1981 tttccccctc gaaaaggtgt caaactactg ccgcataaac tacggttatc ccaaatttaa
     2041 gagaaagcaa taaagcatca gctagcatgc ctttgtctca agaagaatcc accctcattg
     2101 aaagagcaac ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg
     2161 cagctctctc tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg
     2221 gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga
     2281 cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc
     2341 gacaggtgct tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac
     2401 agccgacggc agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaac
     2461 tcgagtgccc atttagttgg cttttccctt gtctcgtgtc ttttccgtgg aaaggttccc
     2521 ggagtaatct gatggcacag cagggaggtg cgcctgcagg ttggttagga aggggggatg
     2581 atgtaaaaga agaaaatggg gggattcgag ccccgcacag gtctctcaaa ttggatgcat
     2641 agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc atagctgttt
     2701 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     2761 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     2821 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2881 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2941 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3001 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3061 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3121 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3181 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3241 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3301 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3361 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3421 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3481 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     3541 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3601 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3661 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3721 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3781 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3841 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3901 tcatccatag ttgcctgact ccccgtcgtg tagataatta cgatacggga gggcttacca
     3961 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4021 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4081 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4141 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4201 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4261 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4321 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4381 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4441 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4501 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4561 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4621 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4681 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4741 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4801 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg aaataccgca
     4861 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa
     4921 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     4981 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5041 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5101 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5161 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     5221 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     5281 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cacttaatgc gccgctacag
     5341 ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     5401 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     5461 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact
     5521 cactata
//
